During fertilization in mice, zona pellucida glycoprotein ZP3 mediates initial sperm-egg interactions by serving as receptor for sperm. Purified egg ZP3, as well as ZP3-derived O-linked oligosaccharides, exhibit sperm receptor activity in vitro. We report that treatment of purified egg ZP3 and ZP3-derived O-linked oligosaccharides with either alpha-galactosidase or galactose oxidase results in loss of sperm receptor activity. In the latter case, sperm receptor activity can be restored to the oxidized glycoprotein and O-linked oligosaccharides by treatment with sodium borohydride. We conclude that galactose, located in alpha-linkage at the nonreducing terminus of O-linked oligosaccharides, is at least one of the sugar determinants on ZP3 responsible for binding of sperm to the zona pellucida.